The resistance in Escherichia coli to clinically important antimicrobial agents including fluoroquinolones and third-generation cephalosporins was investigated in the Tama river watershed to obtain the regional prevalence of the resistance to the newer antimicrobials. Among a total of 3,629 isolates, 78 strains (2.1%) were resistant to cefotaxime (probable extended-spectrum β-lactamases producers) and 76 isolates were resistant to levofloxacin, although no carbapenem-resistant strains were found. A high occurrence of the multiple resistant bacteria to different classes of newer antimicrobials was seen. The gap in the resistant ratio on the boundary between the mountainous area and urbanized area suggests that the antimicrobial resistance is a more sensitive indicator for fecal contamination than the density of E. coli. The resistant ratios of the environmental samples taken even at the middle to downstream in the watershed were lower than those of clinical isolates reported in a nationwide monitoring.